Biphasic mixtures of GLP-1 and insulin

ABSTRACT

The present invention encompasses pharmaceutical formulations comprising a biphasic mixture which comprises a glucagon like peptide (GLP-1) compound in a solid phase and an insulin in a solution phase.

This is the national phase application, under 35 USC 371, forPCT/US02/29842 filed Oct. 7, 2002, which claims the priority of U.S.provisional application No. 60/350,676 filed Oct. 19, 2001.

The present invention relates to biphasic mixtures comprising a glucagonlike peptide (GLP-1) solid phase and an insulin solution phase. Thesebiphasic mixtures can be used to treat diseases such as diabetesmellitus.

It has long been the goal of diabetes therapy to administer drugs thatresult in a pattern of insulin secretion that mimics the pattern ofendogenous insulin secretion in normal individuals. The dailyphysiological demand for insulin fluctuates and can be separated intotwo phases: (a) the absorptive phase requiring a pulse of insulin todispose of the meal-related blood glucose surge, and (b) the postabsorptive phase requiring a sustained delivery of insulin to regulatehepatic glucose output for maintaining optimal fasting blood glucose.

Once oral medications fail to adequately control blood glucose in type 2diabetics, it becomes extremely important to achieve near normalglycemic control and thereby minimize the complications associated withdiabetes. When oral medications fail, the only current alternative is totreat patients with insulin that must be dosed and timed with respect tomeal-related glucose excursions and hepatic glucose output duringperiods of fasting so as to effectively normalize glucose while reducingthe risk of hypoglycemia. Control of the absorptive phase involvingdisposal of the meal-related blood glucose surge can be effectivelyachieved with commercially available regular insulin and monomericinsulin analogs. However, control of the absorptive phase involvingdisposal of hepatic glucose output during periods of fasting, especiallybetween meals and during the bedtime hours, is not as effectivelyachieved with these insulins.

Various commercially available insulin formulations with protracted timeactions have been developed to more conveniently treat the postabsorptive phase. However, it is often quite difficult for type 2diabetics to transition from a treatment involving oral medications toone involving injections of insulin that must be carefully administeredto avoid complications such as hypoglycemia between meals and duringbedtime hours. Thus, there is a need for a more convenient therapy witha reduced risk of hypoglycemia for type 2 diabetics.

Glucagon-like peptide-1 (GLP-1) shows great promise as a treatment fortype 2 diabetes especially for those patients no longer able to controlblood glucose with oral medications. GLP-1 polypeptides have a varietyof physiologically significant activities. For example, GLP-1 has beenshown to stimulate insulin release, lower glucagon secretion, inhibitgastric emptying, and enhance glucose utilization. [Nauck, M. A., et al.(1993) Diabetologia 36:741–744; Gutniak, M., et al. (1992) New EnglandJ. of Med. 326:1316–1322; Nauck, M. A., et al., (1993) J. Clin. Invest.91:301–307]. Furthermore, some animal studies suggest that GLP-1 mayactually preserve beta cells, inhibit beta cell apoptosis, and inducebeta cell proliferation. One of the most exciting observations is thatGLP-1 activity is glucose dependent. When levels drop to a certainthreshold level, GLP-1 is not active. Thus, there is no risk ofhypoglycemia associated with treatment involving GLP-1.

A composition of native GLP-1 and insulin has been suggested by VanAntwerp et al. in WO 01/00223. However, Van Antwerp focuses on thermallystable compositions suitable for continuous infusion using a pump. Theusefulness of these compositions by other means of administration islimited because native GLP-1 in solution is cleared extremely fast andhas a half-life on the order of five minutes.

Derivatives of GLP-1 analogs have been disclosed in U.S. Pat. No.6,268,343. These derivatives having a protracted time action, aregenerally taught as a soluble compostion, which optionally includes anantidiabetic agent, including insulin.

However, it was not understood until the present invention whether aninsoluble GLP-1 and an insulin solution could be formulated togethersuch that both agents are chemically and physically stable and retainthe desired activities and time actions. The molecular interactionsbetween an insoluble precipitate or crystals of GLP-1 in a suspensionand an insulin in solution could compromise the activity and time actionof either agent. Furthermore, the conditions necessary to achievechemical and physical stability are different for each agent whenformulated alone.

The present invention focuses on stable biphasic mixtures that provideoptimal glycemic control with a reduced risk of hypoglycemia. Thebiphasic mixtures comprise a GLP-1 solid phase and an insulin solutionphase. The GLP-1 solid phase comprises an insoluble GLP-1 precipitate orcrystal. The insoluble GLP-1 provides for a slowed absorption rateresulting in GLP-1 with a protracted action that is useful to controldisposal of hepatic glucose output during periods of fasting, especiallybetween meals and during the bedtime hours, as well as meal-relatedblood glucose surges. The insulin solution phase comprises an insulinthat can control disposal of the meal-related blood glucose surge,especially after the first meal of the day where glucose levels arepotentially the highest.

In one form thereof, the present invention provides a pharmaceuticalformulation comprising a biphasic mixture which comprises a glucagonlike peptide (GLP-1) compound in a solid phase and an insulin in asolution phase. Preferably GLP-1 compounds have a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Preferably insulins includeregular human insulin or a monomeric insulin analog. The monomericinsulin analog is preferably selected from the group consisting ofAspB28, AspB28-ProB29, LysB28, LysB28-ProB29, LeuB28, LeuB28-ProB29,ValB28, ValB28-ProB29, AlaB28, AlaB28-ProB29, des B28-30, and des B27.Optionally, the monomeric insulin analog is LysB3-GluB29. Thepharmaceutical formulation can optionally comprise an isotonicity agent.The pharmaceutical formulation can optionally comprise a preservative.

The present invention provides a process of preparing biphasic mixturessuitable for use in pharmaceutical formulations by mixing a GLP-1compound in a solid phase with an insulin in a solution phase, such thatthe GLP-1 remains in a solid phase and retains the sustainedpharmacokinetic profile and the insulin remains in the solution phaseand retains a short time action characteristic.

The present invention provides a method of administering thepharmaceutical formulation administering an effective amount of theformulation comprising a biphasic mixture to a patient in need thereof.

The present invention provides a method of treating a medical conditionselected from the group consisting of non-insulin dependent diabetes,insulin dependent diabetes, hyperglycemia, obesity, catabolic changesafter surgery, myocardial infarction, stress induced hypergycemia, andstroke comprising administering an effective amount of thepharmaceutical formulation comprising a biphasic mixture to a patient inneed thereof.

The present invention provides a use of the pharmaceutical formulationcomprising a biphasic mixture for the preparation of a medicament in thetherapeutic treatment of a medical condition selected from the groupconsisting of non-insulin dependent diabetes, insulin dependentdiabetes, hyperglycemia, obesity, therapeutic reduction of body weightin a human subject, catabolic changes after surgery, myocardialinfarction, stress induced hypergycemia, and stroke in a mammal.

The three-letter abbreviation code for amino acids used in thisspecification conforms with the list contained in Table 3 of Annex C,Appendix 2 of the PCT Administrative Instructions and with 37 C.F.R. §1.822(d)(1)(2000).

For purposes of the present invention as disclosed and described herein,the following terms and abbreviations are defined as follows.

The term “GLP-1 solid” as used herein refers to one phase of a biphasicmixture. The GLP-1 solid phase comprises an insoluble GLP-1 precipitateor crystal in an aqueous solution, wherein the insoluble GLP-1precipitate or crystal has a sustained pharmacokinetic profile. Theinsoluble GLP-1 precipitate or crystal comprises a GLP-1 compound andzinc. Optionally, the insoluble GLP-1 precipitate or crystal furthercomprises a basic polypeptide.

The term “GLP-1” or GLP-1 compound” as used herein refers topolypeptides that include naturally occurring truncated GLP-1polypeptides (GLP-1(7-37)OH and GLP-1(7-36)NH₂), GLP-1 fragments, GLP-1analogs, and derivatives thereof. For purposes of the present invention,GLP-1 compounds also include Exendin-3 and Exendin-4, and analogs andderivatives thereof. GLP-1 compounds of the present invention have theability to bind to the GLP-1 receptor and initiate a signal transductionpathway resulting in insulinotropic activity. Examples of GLP-1compounds appropriate for use in the present invention are discussedmore extensively below.

The term “sustained pharmacokinetic profile” as used herein refers tolength of time efficacious levels of biologically active GLP-1 compoundis in circulation. It is preferable that the sustained pharamacokineticprofile be such that a single injection adequately controls hepaticglucose output during periods of fasting. It is more preferable thatefficacious levels of the GLP-1 compound remain in the serum from about12 hours to about 24 hours, and most preferably from about 20 hours toabout 24 hours.

The term “insulinotropic activity” refers to the ability to stimulateinsulin secretion in response to elevated glucose levels, therebycausing glucose uptake by cells and decreased plasma glucose levels.Insulinotropic activity can be assessed by methods known in the art,including using in vivo experiments and in vitro assays that measureGLP-1 receptor binding activity or receptor activation, e.g., assaysemploying pancreatic islet cells or insulinoma cells, as described in EP619,322 to Gelfand, et al. (described in Example 1), and U.S. Pat. No.5,120,712, respectively. Insulinotropic activity is routinely measuredin humans by measuring insulin levels or C-peptide levels. For thepurposes of the present invention, insulinotropic activity is determinedusing the method described in Example 1. A GLP-1 compound hasinsulinotropic activity if islet cells secrete insulin levels in thepresence of the GLP-1 compound above background levels. Preferably thebiphasic mixtures encompassed by the present invention are comprised ofa GLP-1 compound with insulinotropic activity that is equal to orgreater than GLP-1(7-37)OH. It is even more preferable that the GLP-1compound have greater insulinotropic activity than GLP-1(7-37)OH.

The term “insulin solution” as used herein refers to a second phase in abiphasic mixture. The insulin solution phase comprises an aqueoussolution comprising a soluble insulin, wherein the insulin has a shorttime action characteristic. Preferably, the short time characteristic iscomparable to commercially available insulins, such as Humulin®,Humalog®, Novolog®, and the like. Insulin includes regular insulins,insulin analogs, or insulin derivatives of regular insulins or insulinanalogs that bind to the insulin receptor and initiate the utilizationof circulating glucose. It is preferable that the insulin counteract themeal-related blood glucose surge and return glucose levels back tonormal physiological range. It is more preferable that insulincounteract the meal-related blood glucose surge and return glucoselevels back to normal physiological range within a few hours after ameal. It is more preferable that the insulin counteract the meal-relatedblood glucose surge and return glucose levels back to normalphysiological range within one hour after a meal. It is most preferablethat the insulin counteract the meal-related blood glucose surge afterthe first meal of the day.

Biphasic Mixtures:

The present invention encompasses various biphasic mixtures comprising aGLP-1 compound in a solid phase and an insulin in a solution phase. Thefinal concentrations of the GLP-1 and the insulin in the biphasicmixture will vary depending on the ratios of the two phases. Further,the concentrations will vary depending on the amino acid make-up andpotency of the GLP-1 compound and insulin used. The final concentrationof the GLP-1 in the biphasic mixture is between about 0.1 mg/mL andabout 10 mg/mL. More preferably the final concentration of the GLP-1 isbetween about 0.1 mg/mL and about 8 mg/mL and more preferably betweenabout 0.1 mg/mL and about 7 mg/mL. Most preferably the finalconcentration of the GLP-1 is between about 0.1 mg/mL and about 6 mg/mL.The final insulin concentration is less than about 100 U/mL. (Forexample, 100 U/mL is equal to about 3.5 mg/mL for Humulin® or Humalog®)Preferably the final insulin stock concentration in the biphasic mixtureis less than about 75 U/mL. More preferably the final insulinconcentration is less than 50 U/mL, and most preferably the finalinsulin concentration is about 25 U/mL. The skilled artisan willrecognize that weights (mg) of commercially available insulins, such asHumulin®, Humalog®, and Novolog® are standardized to the number of unitsper milliliter.

Pharmaceutical Formulations:

The present invention encompasses pharmaceutical formulations comprisinga biphasic mixture suitable for administration to a patient in needthereof. Preferably, the pharmaceutical formulation remains stable foran extended period of time under normal conditions of storage.Preferably, the period of time is more than 6 months at 4° C. or ambienttemperature, preferably the period of time is more than 1 year at 4° C.or ambient temperature, more preferably, the period of time is more than2 years at 4° C. or ambient temperature.

The weight to weight ratio of GLP-1 to insulin is such that afteradministration of the pharmaceutical formulation, the plasma levels ofboth the GLP-1 and insulin are maintained within their efficaciousranges. Typically the ratio of GLP-1 to insulin is from about 99:1(weight:weight) to 10:90 (w/w), more preferably, at a ratio from about85:15 (w/w) to 15:85 (w/w) (see Example 6). Even more preferably, theratio of GLP-1 to insulin is from about 85:15 (w/w) to about 50:50(w/w). Most preferably the ratio of GLP-1 to insulin is about 85:15(w/w).

Preferably, serum levels of the GLP-1 that has insulinotropic activitywithin 2-fold that of GLP-1(7-37)OH is maintained between about 30picomoles/liter and about 200 picomoles/liter for at least a timesufficient to control hepatic glucose output during periods of fasting.Optimum serum levels will be higher for GLP-1 compounds that are lessactive than GLP-1(7-37)OH or lower for GLP-1 compounds that are moreactive than GLP-1(7-37)OH. Thus, the concentration of the GLP-1 compoundmay be adjusted upwards or downwards depending on the activity of theGLP-1 compound in the suspension.

Preferably the serum levels of insulin are such as to counteract themeal-related blood glucose surge and restore plasma glucose levels backto normal, typically blood glucose levels are to about 120–125 mg/dL.Generally, the total insulin daily dose is between about 0.3 U/kg andabout 1.5 U/kg. Typically, however, the total mealtime insulin dailydose is between about 50% of the total insulin daily dose, or betweenabout 0.15 U/kg and about 1 U/kg, preferably between about 0.3 U/kg andabout 1 U/kg.

The various pharmaceutical formulations of the present invention mayoptionally encompass a pharmaceutically acceptable buffer. However, theselection, concentration, and pH of the buffer shall be such that theGLP-1 remains in a solid phase and maintains the sustainedpharmacokinetic profile and that the insulin remains in the solutionphase and maintains the short time action characteristic ofcounteracting the meal-related blood glucose surge. Examples ofpharmaceutically acceptable buffers include phosphate buffers such asdibasic sodium phosphate, TRIS, glycylglycine, maleate, sodium acetate,sodium citrate, sodium tartarate, or an amino acid such as glycine,histidine, lysine or arginine. Other pharmaceutically acceptable buffersare known in the art. Preferably, the buffer is selected from the groupconsisting of phosphate, TRIS, maleate, and glycine. Even morepreferably the buffer is TRIS, glycine, or both.

Preferably, the TRIS concentration is between about 1 mM and 100 mM.Even more preferably, the concentration is between about 10 mM and about50 mM, most preferably the buffer is about 15 mM. Preferably, theglycine concentration is between about 10 mM to about 50 mM. Morepreferably, the glycine concentration is between about 20 mM to about 30mM and more highly preferred is a glycine concentration of between about23 mM and about 29 mM.

The pH of the pharmaceutical formulations is adjusted to provideacceptable stability, to maintain the sustained pharmacokinetic profileof the GLP-1 solid and the short time action characteristic of theinsulin solution, and be acceptable for parenteral administration.Preferably, the pH is adjusted to between about 7.0 and about 8.5, morepreferably the pH is between about 7.4 and 8.0, even more preferably thepH is between about 7.4 and 7.8. Most preferably, the pH is about 7.5

The pharmaceutical formulations of the present invention may optionallycomprise a preservative. However, the selection and concentration of thepreservative shall be such that the GLP-1 remains in a solid phase andmaintains sustained pharmacokinetic profile and that the insulin remainsin the solution phase and maintains the short time actioncharacteristic. Preservative refers to a compound that is added to apharmaceutical formulation to act as an anti-microbial agent. Aparenteral formulation must meet guidelines for preservativeeffectiveness to be a commercially viable multi-use product. Amongpreservatives known in the art as being effective and acceptable inparenteral formulations are phenolic preservatives, alkylparabens,benzyl alcohol, chlorobutanol, resorcinol, and other similarpreservatives, and various mixtures thereof. Examples of phenolicderivatives include cresols and phenol or a mixture of cresols andphenol. Examples of cresols include meta-cresol, ortho-cresol,para-cresol, chloro-cresol, or mixtures thereof. Alkylparaben refers toa C₁ to C₄ alkyl paraben, or mixtures thereof. Examples of alkylparabensinclude methylparaben, ethylparaben, propylparaben, or butylparaben. Theconcentrations must be sufficient to maintain preservative effectivenessby retarding microbial growth. Preferably, the preservative is a phenolderivative. More preferably the preservative is a cresol. Even morepreferably the preservative is meta-cresol.

A preferred concentration of a preservative in the final mixture isabout 1.0 mg/mL to about 20.0 mg/mL. More preferred ranges ofconcentration of preservative in the final mixture are about 2.0 mg/mLto about 8.0 mg/mL, about 2.5 mg/mL to about 4.5 mg/mL and about 2.0mg/mL to about 4.0 mg/mL. A most preferred concentration of preservativein the final mixture is about 3.0 mg/mL.

The pharmaceutical formulations of the present invention may optionallycomprise an isotonicity agent. However, the selection and concentrationof the isotonicity agent shall be such that the GLP-1 remains in a solidphase and maintain sustained pharmacokinetic profile and that theinsulin remains in the solution phase and maintains the short timeaction characteristic. Isotonicity agents refer to compounds that aretolerated physiologically and impart a suitable tonicity to theformulation to prevent the net flow of water across cell membranes.Examples of such compounds include glycerin, salts, e.g., NaCl, andsugars, e.g., dextrose, mannitol, and sucrose. These compounds arecommonly used for such purposes at known concentrations. One or moreisotonicity agents may be added to adjust the ionic strength ortonicity. The preferred isotonicity agent is NaCl. The concentration ofthe NaCl is preferably about 110 mM.

A preferred pharmaceutical formulation of the present inventioncomprises a GLP-1 solid phase and a LysB28-ProB29 insulin solution phasesuch as Humalog®. Preferably, GLP-1 to LysB28-ProB29 insulin is at aratio from about 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratiofrom about 85:15 (w/w) to 15:85 (w/w), and even more preferably at aratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises a GLP-1solid phase and an AspB28 insulin solution phase such as Novolog®.Preferably, GLP-1 to AspB28 insulin is at a ratio from about at a ratiofrom about 99:1 (w/w) to 10:90 (w/w) more preferably, at a ratio fromabout 85:15 (w/w) to 15:85 (w/w), and even more preferably at a ratiofrom about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises a GLP-1solid phase and a regular human insulin solution phase such as Humulin®.Preferably, GLP-1 to regular human insulin is at a ratio from about at aratio from about 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratiofrom about 85:15 (w/w) to 15:85 (w/w), and even more preferably at aratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises a GLP-1solid phase and a LysB3-GluB29 insulin solution phase. Preferably, GLP-1to LysB3-GluB29 insulin is at a ratio from about at a ratio from about99:1 (w/w) to 10:90 (w/w) more preferably, at a ratio from about 85:15(w/w) to 15:85 (w/w), and even more preferably at a ratio from about85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises aVal⁸-GLP-1 solid phase and a LysB28-ProB29 insulin solution phase suchas Humalog®. Preferably, Val⁸-GLP-1 to LysB28-ProB29 insulin is at aratio from about at a ratio from about 99:1 (w/w) to 10:90 (w/w), morepreferably, at a ratio from about 85:15 (w/w) to 15:85 (w/w), and evenmore preferably at a ratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises aVal⁸-GLP-1 solid phase and an AspB28 insulin solution phase such asNovolog®. Preferably, Val⁸-GLP-1 to AspB28 insulin is at a ratio fromabout 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratio from about85:15 (w/w) to 15:85 (w/w), and even more preferably at a ratio fromabout 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises aVal⁸-GLP-1 solid phase and a regular human insulin solution phase suchas Humulin®. Preferably, Val⁸-GLP-1 to regular human insulin is at aratio from about 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratiofrom about 85:15 (w/w) to 15:85 (w/w), and even more preferably at aratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises eitherExendin-3 solid phase or Exendin-4 solid phase and LysB28-ProB29 insulinsolution phase such as Humalog®. Preferably, Exendin-3 or Exendin-4 toLysB28-ProB29 insulin is at a ratio from about 99:1 (w/w) to 10:90(w/w), more preferably, at a ratio from about 85:15 (w/w) to 15:85(w/w), and even more preferably at a ratio from about 85:15 (w/w) toabout 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises eitherExendin-3 solid phase or Exendin-4 solid phase and an AspB28 insulinsolution phase such as Novolog®. Preferably, Exendin-3 or Exendin-4 toAspB28 insulin is at a ratio from about 99:1 (w/w) to 10:90 (w/w), morepreferably, at a ratio from about 85:15 (w/w) to 15:85 (w/w), and evenmore preferably at a ratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises eitherExendin-3 solid phase or Exendin-4 solid phase and a regular humaninsulin solution phase such as Humulin®. Preferably, Exendin-3 orExendin-4 to regular human insulin is at a ratio from about 99:1 (w/w)to 10:90 (w/w), more preferably, at a ratio from about 85:15 (w/w) to15:85 (w/w), and even more preferably at a ratio from about 85:15 (w/w)to about 50:50 (w/w).

In another embodiment, the pharmaceutical formulation comprises eitherExendin-3 solid phase or Exendin-4 solid phase and a LysB3-GluB29insulin solution phase. Preferably, GLP-1 to LysB3-GluB29 insulin is ata ratio from about at a ratio from about 99:1 (w/w) to 10:90 (w/w) morepreferably, at a ratio from about 85:15 (w/w) to 15:85 (w/w), and evenmore preferably at a ratio from about 85:15 (w/w) to about 50:50 (w/w).

Administration may be via any route known to be effective by thephysician of ordinary skill. Preferably, the pharmaceutical formulationsof the present invention are administrated parenterally. Parenteraladministration includes intramuscular, subcutaneous, intravenous,intraderamal, and intraperitoneal administration routes. Intramuscularand subcutaneous administration routes are more preferred.

Preferably, when injected, the pharmaceutical formulations of thepresent invention result in a glucose profile that is the same or betterthan that obtained when the GLP-1 solid in a suspension and insulinsolution are administered separately. For example, an injection of thepharmaceutical formulation will result in HbA1_(c) levels that are thesame or lower than the HbA1_(c) levels observed when the GLP-1 solid ina suspension and insulin solution are administered separately. Also, aninjection of the pharmaceutical formulation will preferably mimic thepattern of endogenous insulin secretion in normal individuals. Morepreferably, when injected, the pharmaceutical formulation will result ina glucose profile that is better than that obtained when the GLP-1 solidin a suspension and insulin solution are administered separately.

The pharmaceutical formulations of the present invention are suitable totreat a disease or condition wherein the physiological effects ofadministering GLP-1 or insulin improves the disease or condition.

Included are subjects with non-insulin dependent diabetes, insulindependent diabetes, stroke (see WO 00/16797 by Efendic), myocardialinfarction (see WO 98/08531 by Efendic), obesity (see WO 98/19698 byEfendic), catabolic changes after surgery (see U.S. Pat. No. 6,006,753to Efendic), functional dyspepsia and irritable bowel syndrome (see WO99/64060 by Efendic). Also included are subjects requiring prophylactictreatment with a basal GLP-1 compound, e.g., subjects at risk fordeveloping non-insulin dependent diabetes (see WO 00/07617). Additionalsubjects include those with impaired glucose tolerance or impairedfasting glucose, subjects whose body weight is about 25% above normalbody weight for the subject's height and body build, subjects with apartial pancreatectomy, subjects having one or more parents withnon-insulin dependent diabetes, subjects who have had gestationaldiabetes and subjects who have had acute or chronic pancreatitis are atrisk for developing non-insulin dependent diabetes.

The pharmaceutical formulations of the present invention can be used tonormalize blood glucose levels, prevent pancreatic β-cell deterioration,induce β-cell proliferation, stimulate insulin gene transcription,up-regulate IDX-1/PDX-1 or other growth factors, improve β-cellfunction, activate dormant β-cells, differentiate cells into β-cells,stimulate β-cell replication, inhibit β-cell apoptosis, regulate bodyweight, and induce weight loss.

The pharmaceutical formulations of the present invention preferably havea sustained pharamacokinetic profile that lasts from about 12 hours toabout 24 hours, and most preferably have a sustained pharamacokineticprofile that lasts from about 20 hours to about 24 hours. Thus, a methodof administering the pharmaceutical formulations of the presentinvention involves administration of the appropriate dose twice per daybefore the morning and evening meals, and most preferably once per daybefore the morning meal.

GLP-1 Compounds:

The GLP-1 compounds of the present invention can be made by a variety ofmethods known in the art such as solid-phase synthetic chemistry,purification of GLP-1 molecules from natural sources, recombinant DNAtechnology, or a combination of these methods. For example, methods forpreparing GLP-1 peptides are described in U.S. Pat. Nos. 5,118,666,5,120,712, 5,512,549, 5,977,071, and 6,191,102.

The GLP-1 compounds useful in the present invention include thenaturally occurring truncated GLP-1 polypeptides (GLP-1(7-37)OH andGLP-1(7-36)NH₂), GLP-1 analogs, Exendin 3, and Exendin-4.

The two naturally occurring truncated GLP-1 peptides are represented informula I, SEQ ID NO: 1.

7   8   9   10  11  12  13  14  15  16  17His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-29  30  31  32  33  34  35  36  37 Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaawherein:Xaa at position 37 is Gly, or —NH₂.

Preferably, a GLP-1 compound has the amino acid sequence of SEQ ID NO: 1or is modified so that from one, two, three, four or five amino acidsdiffer from SEQ ID NO:1.

Some GLP-1 compounds known in the art include, for example, GLP-1(7-34)and GLP-1(7-35), GLP-1(7-36), Gln⁹-GLP-1(7-37), D-Gln⁹-GLP-1(7-37),Thr¹⁶-Lys¹⁸-GLP-1(7-37), and Lys¹⁸-GLP-1(7-37). GLP-1 compounds such asGLP-1(7-34) and GLP-1(7-35) are disclosed in U.S. Pat. No. 5,118,666.Other known biologically active GLP-1 analogs are disclosed in U.S. Pat.Nos. 5,977,071; 5,545,618; 5,705,483; 5,977,071; 6,133,235; Adelhorst,et al., J. Biol. Chem. 269:6275 (1994); and Xiao, Q., et al. (2001),Biochemistry 40:2860–2869.

GLP-1 compounds also include polypeptides in which one or more aminoacids have been added to the N-terminus and/or C-terminus ofGLP-1(7-37)OH, or fragments or analogs thereof. Preferably from one tosix amino acids are added to the N-terminus and/or from one to eightamino acids are added to the C-terminus of GLP-1(7-37)OH. It ispreferred that GLP-1 compounds of this type have up to about thirty-nineamino acids. The amino acids in the “extended” GLP-1 compounds aredenoted by the same number as the corresponding amino acid inGLP-1(7-37)OH. For example, the N-terminal amino acid of a GLP-1compound obtained by adding two amino acids to the N-terminus ofGLP-1(7-37)OH is at position 5 and 6; and the C-terminal amino acid of aGLP-1 compound obtained by adding one amino acid to the C-terminus ofGLP-1(7-37)OH is at position 38. Amino acids 1–6 of an extended GLP-1compound are preferably the same as or a conservative substitution ofthe amino acid at the corresponding position of GLP-1(1-37)OH. Aminoacids 38–45 of an extended GLP-1 compound are preferably the same as ora conservative substitution of the amino acid at the correspondingposition of Exendin-3 or Exendin-4. The amino acid sequence of Exendin-3and Exendin-4 are represented in formula II, SEQ ID NO: 2.

7   8   9   10  11  12  13  14  15  16  17His-Xaa-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-18  19  20  21  22  23  24  25  26  27  28Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-29  30  31  32  33  34  35  36  37  38  39Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser- 40  41  42  43  44  45Gly-Ala-Pro-Pro-Pro-Ser-NH₂wherein:Xaa at position 8 is Ser or Gly; andXaa at position 9 is Asp or Glu.

Exendin-3 has Ser at position 8 and Asp at position 9. Exendin-4 has Glyat position 8 and Glu at position 9. Other GLP-1 compounds of thepresent invention include formula 2 (SEQ ID NO:2) wherein the C-terminalSer is the acid form instead of the amidated form. Also, GLP-1 compoundsof the present invention include Exendin-3 and Exendin-4 agonists asdescribed in WO99/07404, WO99/25727, WO99/25728, WO99/43708, WO00/66629,and US2001/0047084A1 which are herein incorporated by reference.

A preferred group of GLP-1 compounds are represented in formula III (SEQID NO:3):

7   8   9   10  11  12  13  14  15  16  17Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Xaa-Asp-Xaa-Xaa-18  19  20  21  22  23  24  25  26  27  28Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-29  30  31  32  33  34  35  36  37  38  39Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- 40  41  42  43  44  45Xaa-Xaa-Xaa-Xaa-Xaa-Xaawherein:Xaa at position 7 is: L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine or α-methyl-histidine;

Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, orLys; Xaa at position 9 is Glu, Asp, Lys, Thr, Ser, Arg, Trp, Phe, Tyr,or His; Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu,Asp, Arg, His, or Lys; Xaa at position 12 is His, Trp, Phe, or Tyr Xaaat position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp,Trp, His, Phe, or Lys; Xaa at position 17 is Ser, Ala, Gly, Thr, Leu,Ile, Val, Glu, Asp, or Lys; Xaa at position 18 is Ser, Ala, Gly, Thr,Leu, Ile, Val, Glu, Asp, His, Pro, Arg, or Lys; Xaa at position 19 isTyr, Phe, Trp, Glu, Asp, Gly, Gln, Asn, Arg, Cys, or Lys; Xaa atposition 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, or Lys;Xaa at position 21 is Glu, Asp, or Lys; Xaa at position 22 is Gly, Ala,Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa at position 23 is Gln,Asn, Arg, Glu, Asp, His, or Lys; Xaa at position 24 is Ala, Gly, Ser,Thr, Leu, Ile, Val, Arg, Glu, Asp, or Lys; Xaa at position 25 is Ala,Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa at position 26 isLys, Arg, Gln, Glu, Asp, Trp, Tyr, Phe, or His; Xaa at position 27 isGlu, Asp, Ala, His, Phe, Tyr, Trp, Arg, Leu, or Lys; Xaa at position 30is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, His, or Lys; Xaa atposition 31 is Trp, Phe, Tyr, Glu, Asp, Ser, Thr, Arg, or Lys; Xaa atposition 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys; Xaaat position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, Arg, orLys; Xaa at position 34 is Lys, Arg, Glu, Asp, Asn, or His; Xaa atposition 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Arg, Trp,Tyr, Phe, Pro, His, or Lys; Xaa at position 36 is Arg, Lys, Glu, Asp,Thr, Ser, Trp, Tyr, Phe, Gly, or His; Xaa at position 37 is Gly, Ala,Ser, Thr, Leu, Ile, Val, Glu, Asp, His, Lys, Arg, Trp, Tyr, Phe, Pro,Pro-NH₂ or is deleted; Xaa at position 38 is Arg, Lys, Glu, Asp, Ser, orHis, or is deleted; Xaa at position 39 is Arg, Lys, Glu, Asp, Ser, orHis, or is deleted; Xaa at position 40 is Asp, Glu, Gly, or Lys, or isdeleted; Xaa at position 41 is Phe, Trp, Tyr, Glu, Asp, Ala, or Lys, oris deleted; Xaa at position 42 is Ser, Pro, Lys, Glu, or Asp, or isdeleted; Xaa at position 43 is Ser, Glu, Asp, Pro, or Lys, or isdeleted; Xaa at position 44 is Gly, Glu, Asp, Pro, or Lys, or isdeleted; and Xaa at position 45 is Ala, Val, Glu, Asp, Ser, or Lys, orAla-NH₂, Val-NH₂, Glu-NH₂, Asp-NH₂, Ser-NH₂, or Lys-NH₂, or is deleted,or a C-1-6-ester, or amide, or C-1-6- alkylamide, or C-1-6-dialkylamidethereof; provided that when the amino acid at position 37, 38, 39, 40,41, 42, 43, or 44 is deleted, then each amino acid downstream of thatamino acid is also deleted.

A preferred group of GLP-1 compounds are:

HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRG or G-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIDGGPSSGRPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRGSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLVKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGGSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGPGSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGSPSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDAPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPAPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPAS or S-NH2HVEGTFTSDVSSYLEEQAAXEFIAWLIKGGPSSGDAAAS or S-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPSSGAPPPH or H-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPHSSGAPPPS or S-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS or S-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLVKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRGSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGRGSSGAPPPS or S-NH2HVEGTFTSDWSSYLEEQAAKEFIAWLIKGRGSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGRGHSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRGHSSGAPPPS or S-NH2HVEGTFTSDWSSYLEEQAAKEFIAWLIKGGPHSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEEQAAKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDVSWYLEGQAVKEFIAWLIKGGPHSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLINKGGPSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEEQAVKEFIAWLIKGGPHSSGAPPPS or S-NH2HVEGTFTSDWSSYLEEQAVKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEEQAVKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDWSKYLEEQAVKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPRG or G-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPRG or G-NH₂HVEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPS or S-NH₂HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGRPPPS or S-NH₂HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGRPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGKPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGRG or G-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSWGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPGPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPGPSGPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLVKGGPSSGAPPPS or S-NH2

Another preferred group of GLP-1 compounds is represented in formula IV(SEQ ID NO:4):

7   8   9   10  11  12  13  14  15  16  17His-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Lys-Xaa-Phe-29  30  31  32  33  34  35  36  37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-Rwherein:

Xaa at position 8 is Gly, Ala, Val, Leu, Ile, Ser, or Thr; Xaa atposition 22 is Asp, Glu, Gln, Asn, Lys, Arg, Cys, or Cysteic Acid; Xaaat position 23 is His, Asp, Lys, Glu, or Gln; Xaa at position 27 is Ala,Glu, His, Phe, Tyr, Trp, Arg, or Lys; Xaa at position 30 is Glu, Asp,Ser, or His; R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His,-NH₂.

It is also preferable that the GLP-1 compounds of the present inventionhave other combinations of substituted amino acids. The presentinvention encompasses a GLP-1 compound comprising the amino acidsequence of formula V (SEQ ID NO:5)

Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Gln-Ala-Xaa₂₅-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Lys-Gly-Arg-Xaa₃₇wherein:Xaa₇ is: L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine, or α-methyl-histidine;

Xaa₈ is: Ala, Gly, Val, Leu, Ile, Ser, or Thr; Xaa₁₂ is: Phe, Trp, orTyr; Xaa₁₆ is: Val, Trp, Ile, Leu, Phe, or Tyr; Xaa₁₈ is: Ser, Trp, Tyr,Phe, Lys, Ile, Leu, Val; Xaa₁₉ is: Tyr, Trp, or Phe; Xaa₂₀ is: Leu, Phe,Tyr, or Trp; Xaa₂₂ is: Gly, Glu, Asp, Lys; Xaa₂₅ is: Ala, Val, Ile, orLeu; Xaa₂₇ is: Glu, Ile, or Ala; Xaa₃₀ is: Ala or Glu Xaa₃₃ is: Val, orIle; and Xaa₃₇ is: Gly, His, -NH₂, or is absent.

The present invention also encompasses a GLP-1 compound comprising theamino acid sequence of formula VI (SEQ ID NO:6)

Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Leu-Glu-Xaa₂₂-Gln-Ala-Xaa₂₅-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Xaa₃₃-Lys-Gly-Arg- Xaa₃₇wherein:Xaa₇ is: L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine, or α-methyl-histidine;

Xaa₈ is: Gly, Ala, Val, Leu, Ile, Ser, or Thr; Xaa₁₆ is: Val, Phe, Tyr,or Trp; Xaa₁₈ is: Ser, Tyr, Trp, Phe, Lys, Ile, Leu, or Val; Xaa₂₂ is:Gly, Glu, Asp, or Lys; Xaa₂₅ is: Ala, Val, Ile, or Leu; Xaa₃₃ is: Val orIle; and Xaa₃₇ is: Gly, —NH₂, or is absent.

Most preferred GLP-1 compounds comprise GLP-1 analogs wherein thebackbone for such analogs or fragments contains an amino acid other thanalanine at position 8 (position 8 analogs). Preferred amino acids atposition 8 are glycine, valine, leucine, isoleucine, serine, threonine,or methionine and more preferably are valine or glycine.

Other preferred GLP-1 compounds are GLP-1 analogs that have the sequenceof GLP-1(7-37)OH except that the amino acid at position 8 is preferablyglycine, valine, leucine, isoleucine, serine, threonine, or methionineand more preferably valine or glycine and position 22 is glutaric acid,lysine, aspartic acid, or arginine and more preferably glutamic acid orlysine.

Other preferred GLP-1 compounds are GLP-1 analogs that have the sequenceof GLP-1(7-37)OH except that the amino acid at position 8 is preferablyglycine, valine, leucine, isoleucine, serine, threonine, or methionineand more preferably valine or glycine and position 30 is glutamic acid,aspartic acid, serine, or histidine and more preferably glutamic acid.

Other preferred GLP-1 compounds are GLP-1 analogs that have the sequenceof GLP-1(7-37)OH except that the amino acid at position 8 is preferablyglycine, valine, leucine, isoleucine, serine, threonine, or methionineand more preferably valine or glycine and position 37 is histidine,lysine, arginine, threonine, serine, glutamic acid, aspartic acid,tryptophan, tyrosine, phenylalanine and more preferably histidine.

Other preferred GLP-1 compounds are GLP-1 analogs that have the sequenceof GLP-1(7-37)OH except that the amino acid at position 8 is preferablyglycine, valine, leucine, isoleucine, serine, threonine, or methionineand more preferably valine or glycine and position 22 is glutamic acid,lysine, aspartic acid, or arginine and more preferably glutamic acid orlysine and position 27 is alanine, lysine, arginine, tryptophan,tyrosine, phenylalanine, or histidine and more preferably alanine.

In the nomenclature used herein to describe GLP-1 compounds, thesubstituting amino acid and its position is indicated prior to theparent structure. For example Val⁸-GLP-1(7-37)OH designates a GLP-1compound in which the alanine normally found at position 8 inGLP-1(7-37)OH (formula I, SEQ ID NO:1) is replaced with valine.

Other preferred GLP-1 compounds include: Val⁸-GLP-1(7-37)OH,Gly⁸-GLP-1(7-37)OH, Glu²²-GLP-1(7-37)OH, Asp²²-GLP-1(7-37)OH,Arg²²-GLP-1(7-37)OH, Lys²²-GLP-1(7-37)OH, Cys²²-GLP-1(7-37)OH,Val⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Asp²²-GLP-1(7-37)OH,Val⁸-Arg²²-GLP-1(7-37)OH, Val⁸-Lys²²-GLP-1(7-37)OH,Val⁸-Cys²²-GLP-1(7-37)OH, Gly⁸-Glu²²-GLP-1(7-37)OH,Gly⁸-Asp²²-GLP-1(7-37)OH, Gly⁸-Arg²²-GLP-1(7-37)OH,Gly⁸-Lys²²-GLP-1(7-37)OH, Gly⁸-Cya²²-GLP-1(7-37)OH,Glu²²-GLP-1(7-36)NH₂, Asp²²-GLP-1(7-36)NH₂, Arg ²²-GLP-1(7-36)NH₂,Lys²²-GLP-1 (7-36)NH₂, Cys²²-GLP-1 (7-36)NH₂, Val⁸-Glu²²-GLP-1(7-36)NH₂,Val⁸-Asp²²-GLP-1(7-36)NH₂, Val⁸-Arg²²-GLP-1(7-36)NH₂,Val⁸-Lys²²-GLP-1(7-36)NH₂, Val⁸-Cys²²-GLP-1(7-36)NH₂,Gly⁸-Glu²²-GLP-1(7-36)NH₂, Gly⁸-Asp²²-GLP-1(7-36)NH₂,Gly⁸-Arg²²-GLP-1(7-36)NH₂, Gly⁸-Lys²²-GLP-1(7-36) NH₂,Gly⁸-Cys²²-GLP-1(7-36)NH₂, Lys²³-GLP-1(7-37)OH, Val⁸-Lys²³-GLP-1(7-37)OH, Gly⁸-Lys²³-GLP-1(7-37)OH, His²⁴-GLP-1(7-37)OH,Val⁸-His²⁴-GLP-1(7-37)OH, Gly ⁸-His²⁴-GLP-1(7-37)OH,Lys²⁴-GLP-1(7-37)OH, Val⁸-Lys²⁴-GLP-1(7-37)OH, Gly⁸-Lys²³-GLP-1(7-37)OH, Glu³⁰-GLP-1(7-37)OH, Val⁸-Glu³⁰-GLP-1(7-37)OH,Gly ⁸-Glu³⁰-GLP-1(7-37)OH, Asp³⁰-GLP-1(7-37)OH,Val⁸-Asp³⁰-GLP-1(7-37)OH, Gly ⁸-Asp³⁰-GLP-1(7-37)OH,Gln³⁰-GLP-1(7-37)OH, Val⁸-Gln³⁰-GLP-1(7-37)OH, Gly⁸-Gln³⁰-GLP-1(7-37)OH, Tyr³⁰-GLP-1(7-37)OH, Val⁸-Tyr³⁰-GLP-1(7-37)OH,Gly ⁸-Try³⁰-GLP-1(7-37)OH, Ser³⁰-GLP-1(7-37)OH,Val⁸-Ser³⁰-GLP-1(7-37)OH, Gly ⁸-Ser³⁰-GLP-1(7-37)OH,His³⁰-GLP-1(7-37)OH, Val⁸-His³⁰-GLP-1(7-37)OH, Gly⁸-His³⁰-GLP-1(7-37)OH, Glu³⁴-GLP-1(7-37)OH, Val⁸-Glu³⁴-GLP-1(7-37)OH,Gly ⁸-Glu³⁴-GLP-1(7-37)OH, Ala³⁴-GLP-1(7-37)OH,Val⁸-Ala³⁴-GLP-1(7-37)OH, Gly ⁸-Ala³⁴-GLP-1(7-37)OH,Gly³⁴-GLP-1(7-37)OH, Val⁸-Gly³⁴-GLP-1(7-37)OH, Gly⁸-Gly³⁴-GLP-1(7-37)OH, Ala³⁵-GLP-1(7-37)OH, Val⁸-Ala³⁵-GLP-1(7-37)OH,Gly ⁸-Ala³⁵-GLP-1(7-37)OH, Lys³⁵-GLP-1(7-37)OH,Val⁸-Lys³⁵-GLP-1(7-37)OH, Gly ⁸-Lys³⁵-GLP-1(7-37)OH,His³⁵-GLP-1(7-37)OH, Val⁸-His³⁵-GLP-1(7-37)OH, Gly⁸-His³⁵-GLP-1(7-37)OH, Pro³⁵-GLP-1(7-37)OH, Val⁸-Pro³⁵-GLP-1(7-37)OH,Gly⁸-Pro³⁵-GLP-1(7-37)OH, Glu³⁵-GLP-1 (7-37)OH Val⁸-Glu³⁵-GLP-1(7-37)OH,Gly⁸-Glu³⁵-GLP -1(7-37)OH, Val⁸-Ala²⁷-GLP-1(7-37)OH,Val⁸-His³⁷-GLP-1(7-37)OH, Val⁸-Glu²²Lys²³-GLP-1(7-37)OH,Val⁸-Glu²²-Glu²³-GLP-1(7-37)OH, Val⁸-Glu²²-Ala²⁷-GLP-1(7-37)OH,Val⁸-Gly³⁴-Lys³⁵-GLP-1(7-37)OH, Val⁸-His³⁷-GLP-1(7-37)OH, andGly⁸-His³⁷-GLP-1(7-37)OH.

More preferred GLP-1 compounds are Val⁸-GLP-1(7-37)OH,Gly⁸-GLP-1(7-37)OH, Glu²²-GLP-1(7-37)OH, Lys²²-GLP-1(7-37)OH,Val⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Lys²²-GLP-1(7-37)OH,Gly⁸-Glu²²-GLP-1(7-37)OH, Gly⁸-Lys²²-GLP-1(7-37)OH,Glu²²-GLP-1(7-36)NH₂, Lys²²-GLP-1(7-36)NH₂, Val⁸-Glu²²-GLP-1(7-36)NH₂,Val⁸-Lys²²-GLP-1(7-36)NH₂, Gly⁸-Glu²²-GLP-1(7-36) NH₂,Gly⁸-Lys²²-GLP-1(7-36)NH₂, Val⁸-His³⁷-GLP-1(7-37)OH,Gly⁸-His³⁷-GLP-1(7-37)OH, Arg³⁴-GLP-1(7-36)NH₂, and Arg³⁴-GLP-1(7-37)OH.

Other preferred GLP-1 compounds include: Val⁸-Tyr¹²-GLP-1(7-37)OH,Val⁸-Tyr¹²-GLP-1(7-36)NH₂, Val⁸-Trp¹²-GLP-1(7-37)OH,Val⁸-Leu¹⁶-GLP-1(7-37)OH, Val⁸-Tyr¹⁶-GLP-1(7-37)OH,Gly⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Leu²⁵-GLP-1(7-37)OH,Val⁸-Glu³⁰-GLP-1(7-37)OH, Val⁸-His³⁷-GLP-1(7-37)OH,Val⁸-Tyr¹²-Tyr¹⁶-GLP-1(7-37)OH, Val⁸-Trp¹²-Glu²²-GLP-1 (7-37)OH,Val⁸-Tyr¹²-Glu²²-GLP-1(7-37)OH, Val⁸-Tyr¹⁶-Phe¹⁹-GLP-1(7-37)OH,Val⁸-Tyr¹⁶-Glu²²-GLP-1(7-37)OH, Val⁸-Trp¹⁶-Glu²²-GLP-1(7-37)OH,Val⁸-Leu¹⁶-Glu²²-GLP-1 (7-37)OH, Val⁸-Ile¹⁶Glu²²-GLP-1(7-37)OH,Val⁸-Phe¹⁶-Glu²²-GLP-1(7-37)OH, Val⁸-Trp¹⁸-Glu²²-GLP-1(7-37)OH,Val⁸-Tyr¹⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Phe¹⁹-Glu^(22-GLP-)1(7-37)OH,Val⁸-Ile¹⁸Glu²²-GLP-1(7-37)OH, Val⁸-Lys¹⁸-Glu²²-GLP-1(7-37)OH,Val⁸-Trp¹⁹-Glu²²-GLP-1(7-37)OH, Val⁸-Phe¹⁹-Glu²²-GLP-1 (7-37)OH,Val⁸-Phe²⁰-Glu²²-GLP-1(7-37)OH, Val⁸-Glu²²-Leu²⁵-GLP-1(7-37)OH,Val⁵-Glu²²-Ile²⁵-GLP-1(7-37)OH, Val⁸-Glu²²-Val²⁵-GLP-1(7-37)OH,Val⁸-Glu²²-Ile²⁷-GLP-1 (7-37)OH, Val⁸-Glu²²-Ala²⁷-GLP-1(7-37)OH,Val⁸-Glu²²-Ile³³-GLP-1(7-37)OH, Val⁸-Glu²²-His³⁷-GLP-1(7-37)OH,Val⁸-Asp¹⁹-Ile¹¹-Tyr¹⁶-Glu²²-GLP-1(7-37)OH, Val⁸-Tyr¹⁶-Trp¹⁹-Glu²²-GLP-1(7-37)OH, Val⁸-Trp¹⁶-Glu ²²-Val²⁵Ile³³-GLP-1(7-37)OH,Val⁸-Trp¹⁶-Glu²²-Ile³³-GLP-1(7-37)OH, Val⁸-Glu²²-Val²²-Val²⁵Ile³³-GLP-37)OH, and Val⁸-Trp¹⁶-Glu²²-Val²⁵-GLP-1(7-37)OH.

Zinc Crystals:

A GLP-1 compound can be incorporated into zinc crystals. Preferably, analkaline normalization step is performed. The pH of the GLP-1 solutionis adjusted to between about 9.5 and about 11.5. This step appears toreduce the content of β-sheet conformation in the peptide and enhancethe α-helix conformation that is important for solubility andbioavailability of some GLP-1 compounds. This step also serves tomaintain the peptide in a preferred α-helix conformation prior to thesubsequent process step. This key step thus “normalizes” variation inbulk lots of the peptide into a more reproducible, homogenous solution.

Preferably, the peptide concentration in the alkaline normalizationsolution is greater than 5 mg/mL. More preferably, the concentration isabout 10 mg/mL to about 30 mg/mL. Other ranges of preferredconcentration of dissolved peptide are about 5 mg/mL to about 25 mg/mL,about 8 mg/mL to about 25 mg/mL and about 10 mg/mL to about 20 mg/mL.The most preferred concentration is about 15 mg/mL.

Preferably, an aqueous alkaline solution comprising only water and abase such as NaOH, KOH or ammonium hydroxide is employed to dissolve thepeptide. A more preferred base is NaOH.

Preferably, the pH of the alkaline normalization step is about 10.0 toabout 11.0. More preferably, the pH is about 10.5. The alkaline solutioncomprising the dissolved peptide may be allowed to sit quiescently for aperiod of about 5 minutes to about 3 hours at ambient temperature,which, although it is not to be construed as a limitation, is generallybetween about 20° C. and about 25° C. The alkaline solution may also begently stirred. More preferably, the dissolved alkaline peptide solutionwill sit quiescently for about 1 hour at ambient temperature. Oneskilled in the art will recognize that combinations of pH, time,temperature and stirring conditions for this step can be readilyestablished for each peptide that ensures “normalization” of the peptideconformation is complete yet avoids or minimizes chemical degradationthat may occur to the peptide.

The next step in the process for preparing crystals of a selectedpeptide is the addition of glycine. Amino acids such as glycine bindzinc ions which also bind very tightly to the histidine residue(s) in apeptide. Thus, competition for zinc binding may play a role in theformation of peptide crystals, as well as in the stability of subsequentcrystalline compositions. The glycine added to the alkaline peptidesolution may be in a solid form or in a stock solution. Preferably,glycine is added as a solid. Preferably, the added glycine is infree-base form. Preferably, the resulting concentration of glycine inthe alkaline peptide solution is about 5 mM to about 250 mM. Ranges ofmore preferred glycine concentration are about 10 mM to about 150 mM,about 20 mM to about 100 mM, about 40 mM to about 80 mM and about 55 mMto about 65 mM. Most preferably, the glycine concentration is about 60mM.

Optionally, the pH of the alkaline peptide solution may be readjustedafter the addition of the glycine. If the pH is adjusted, it ispreferably adjusted to a pH between about 9.0 and about 11.0. Morepreferably, it is adjusted to a pH between about 9.2 and about 9.8. Mostpreferably, it is adjusted to about pH 9.5.

Optionally, the alkaline peptide solution with added glycine may befiltered. Filtration is recommended if any evidence of undissolvedparticles, dust or lint is apparent in the solution. If desired, this isalso a good place in the process at which the solution can be sterilizedby performing an aseptic filtration step. Preferably, the filtrationwill be conducted using a sterile non-pyrogenic filter havinglow-protein binding and a pore size of 0.45 μm or less. Preferably, thefilter is a sterile non-pyrogenic, low-protein binding filter of poresize 0.22 μm or less. More preferably, the filter is a sterile 0.22 μmMillex® filter (Millipore Corporation, Waltham, Mass., USA).

The next step in the process of forming crystals is addition to thealkaline peptide solution of about 2% to about 20% of the total finalvolume of an alcohol selected from the group consisting of ethanol andisopropanol, and about 0.5 moles to about 2.5 moles of zinc per mole ofthe peptide. The zinc and ethanol may be added in a single aqueous stocksolution or may be added separately in one or more steps in any order.Preferably, the alcohol is added before the zinc is added.

Preferably, the added alcohol represents, by volume, about 2% to about20% of the total final volume of the alkaline peptide-zinc-alcoholsolution. More preferably, the alcohol represents about 5% to about 15%of the total final volume. More preferably, the alcohol represents about6% to about 12% of the total final volume. Most preferably, the alcoholrepresents about 9% of the total final volume. Preferably, the alcoholis ethanol.

The zinc added at this stage refers to the zinc ion. The zinc may beadded in a variety of forms, but a zinc oxide solution acidified withdilute HCl and salt forms such as zinc acetate or zinc chloride arepreferred. More preferred is a zinc oxide solution acidified with diluteHCl.

Preferably, 1.0 moles to about 2.25 moles of zinc per mole of thepeptide is added in this process step. Other preferred ranges of zincaddition include 1.1 to 2.0 moles of zinc per mole of the peptide, 1.3to 1.7 moles per mole of peptide, and 1.4 to 1.6 moles per mole ofpeptide. Most preferably, about 1.5 moles of zinc per mole of peptide isadded.

Preferably, the solution comprising zinc that is added to the peptidesolution is added slowly and/or in small increments, which minimizes thelocalized precipitation of peptide and/or zinc complexes that may format the site of addition. More preferably, glycine is also a component ofthe solution comprising zinc that is being added at this step. Forexample, a zinc-glycine solution may be prepared by dissolving zincoxide in dilute HCl to a pH of about 1.6 and then adding solid glycine.A sufficient quantity of glycine is added to raise the pH of thesolution to between about pH 2 and about pH 3. The pH of thezinc-glycine solution may be raised further using, for example, diluteNaOH. A preferred pH range of the zinc-glycine solution is about pH 4.0to about pH 6.0. A more preferred pH range of the zinc-glycine solutionis about pH 5.0 to about pH 5.5. As noted earlier, glycine has a bindingaffinity for zinc that may compete with zinc binding to the peptide.Thus, the presence of glycine in the solution comprising zinc that isbeing added to the composition allows the zinc solution to be added morequickly because localized precipitation problems are minimized. Inaddition, having a zinc-glycine solution above pH 2.0, and preferablyabout pH 4.0 to about pH 6.0, allows the solution to be sterile filteredusing filters that are rated by their manufacturers to handle, forexample, pH 2–10 solutions, prior to its introduction into a sterilepeptide composition. Preferably, the zinc-glycine solution comprisesabout 50 mM to about 70 mM glycine and about 20 mM to about 200 mM zinc.

The last steps in the initial crystallization of a selected peptide areadjusting the pH of the solution to between about pH 7.5 and about pH10.5 and allowing crystals of the peptide to form. Preferred reagentsolutions useful for adjusting the pH of the solution include diluteHCl, dilute acetic acid and dilute NaOH.

Preferred pH ranges for crystallization of selected peptides includeabout pH 8.0 to about pH 10.0, about pH 7.5 to about pH 9.5, about pH8.5 to about pH 9.2, about pH 9.0 to about pH 9.5, about pH 7.5 to aboutpH 8.5, about pH 8.7 to about pH 9.5, and about pH 9.2 to about pH 10.0.

One skilled in the art will recognize that the preferred pH ofcrystallization will depend on many factors, including the nature of thepeptide and its concentration, the alcohol concentration, the zincconcentration, the ionic strength of the solution and the temperature ofcrystallization. By way of illustration, the peptideVal⁸-Glu³⁰-GLP-1(7-37)OH produced crystals at only select ethanol andzinc concentrations at a pH range of about 7.7 to about 8.1, whereas thepeptide Val⁸-His³⁷-GLP-1(7-37)OH produced crystals over a broad range ofzinc and ethanol concentrations at a pH range of about 9.8 to about10.4.

The skilled artisan will further recognize that, for a given set ofconditions, a preferred manner of determining the optimal pH ofcrystallization is to determine it empirically, that is, to slowly addthe acidification solution, preferably dilute HCl or dilute acetic acid,in small increments, and observe what happens after each increment isadded. Generally, small quantities of localized zones of precipitationwill occur at the spot of addition of the acidic solution. When gentleswirling takes increasingly longer periods of time to completelyredissolve the precipitation, that is the best time to stop adding theacid and allow crystallization from the clear or slightly cloudysolution to proceed.

The skilled artisan will further recognize that the pH and temperaturethat one selects for crystallization will have an impact on the speed atwhich the crystallization proceeds, the crystallization yield, and thesize and homogeneity of the crystals formed. Preferably, the pH ofcrystallization for the selected peptides is about pH 8.0 to about pH10. More preferably, the pH is about 8.7 to about 9.5. Other ranges ofpreferred pH of crystallization are about 8.8 to about 9.3, about 9.0 toabout 9.5, and about 8.5 to about 9.3. Most preferably, thecrystallization is conducted at about pH 9.1.

Preferably, the temperature of crystallization is about 10° C. to about30° C. More preferably, the temperature of crystallization is about 15°C. to about 28° C. Most preferably, the temperature of crystallizationis ambient temperature, or about 20° C. to about 25° C.

Preferably, the crystallization step described above is complete, thatis, 90% or more of the peptide is precipitated in predominantlycrystalline form, in about 3 hours to about 72 hours. More preferably,the crystallization is complete in about 10 hours to about 48 hours.Most preferably, the crystallization is complete in about 16 hours toabout 26 hours. Completion of crystallization may be determined by avariety of means, including HPLC analysis of the peptide present in analiquot of the composition. Method 5 herein describes one such protocolthat may be employed. Preferably, the crystals produced according to thesteps of the process described above are thin plate crystals. Thecrystals produced by the process may be examined by microscopy.

The pH of the suspension of crystals in the original mother liquor islowered to a pH value at which 97% or more of the peptide becomesinsoluble. Preferably, this part of the process begins within a fewhours after the initial crystallization is determined to be complete.Preferably, the pH is lowered using a dilute solution of HCl or aceticacid wherein the acidic solution is added slowly and in incrementalportions. The skilled artisan will recognize that the preferred pH atwhich this second stage of crystallization should occur will depend onmany factors, including the nature of the peptide and its concentration,the alcohol concentration, the zinc concentration, the ionic strength ofthe suspension and the temperature of crystallization. Preferably, thepH is about 0.2 to 2.0 pH units lower than the pH at which the initialcrystallization proceeded. More preferably, the pH is about 0.5 to about1.5 pH units lower, and most preferably, the pH is about 0.8 to 1.3 pHunits lower than the pH at which the initial crystallization proceeded.The temperature of this second stage of crystallization is preferablyambient temperature, or about 20° C. to about 25° C. For the peptideVal⁸-GLP-1(7-37)OH, a preferred pH is about 7.5 to about 8.5. A morepreferred pH is about 7.8 to about 8.2.

Preferably, the pH of a suspension of peptide crystals is lowered to apH at which 98% or more, and more preferably at which 99% or more of thepeptide becomes insoluble in the composition. The additionalprecipitation formed in this second stage of crystallization comprisescrystals. Preferably, the additional precipitation formed in this secondstage of crystallization will be predominantly crystals of comparablemorphology and size distribution as those formed in the first stage ofcrystallization.

Preferably, the second stage of crystallization is complete enough, thatis, 97% or more of the peptide is insoluble, to allow the following stepto begin within 30 hours, more preferably within 18 hours, morepreferably within 6 hours and most preferably within 2 hours of when thesecond stage of crystallization started. Quantitation of precipitationyield may be determined by a variety of means, including HPLC analysisof the peptide present in an aliquot of the suspension.

The steps as described above will result in a stock suspensioncomprising an insoluble GLP-1 precipitate or crystals in the originalmother liquor from the initial crystallization stage. The stocksuspension can be mixed with an insulin solution in its present form orthe stock suspension may optionally include other suitable,pharmaceutically acceptable excipients.

Optionally, the stock GLP-1 suspension may include a pharmaceuticallyacceptable buffer such as TRIS, maleate, phosphate, succinate,glycylglycine or adipate, and one or more tonicity agents such as sodiumchloride, other salts, glycerin or mannitol. These components may beadded as a single solution, as combination solutions or individually inany order. Of these components, a preferred buffer is selected from thegroup consisting of TRIS, maleate and glycylglycine, and a preferredtonicity agent is sodium chloride. A more preferred buffer is TRIS.

A preferred quantity of TRIS to add to the stock GLP-1 suspension, ifTRIS is the selected buffer, is such that the TRIS concentration in thefinal composition is about 5 mM to about 40 mM. A more preferred rangeof TRIS concentration in the final composition is about 10 mM to about20 mM. A most preferred concentration of TRIS in the final compositionis about 15 mM.

A preferred quantity of maleate to add to the stock GLP-1 suspension, ifmaleate is the selected buffer, is such that the maleate concentrationin the final composition is about 2 mM to about 20 mM. A more preferredrange of maleate concentration in the final composition is about 5 mM toabout 15 mM. A most preferred concentration of maleate in the finalcomposition is about 9 mM.

If sodium chloride is selected to be a component of the stock GLP-1suspension peptide composition, a preferred quantity to add is such thatthe added sodium chloride in the stock suspension is about 30 mM toabout 200 mM. A more preferred concentration of added sodium chloride inthe stock suspension is 50 mM to about 150 mM. Other ranges of preferredsodium chloride concentration are about 80 mM to about 120 mM, about 70mM to about 130 mM, and about 90 mM to about 130 mM. A most preferredquantity of added sodium chloride in the stock suspension is about 110mM.

Although any pharmaceutically acceptable preservative may be added tothe stock GLP-1 suspension at this point in the process, a phenolicpreservative or benzyl alcohol is preferred. Examples of phenolicpreservatives include phenol, chlorocresol, m-cresol, o-cresol,p-cresol, ethylparaben, methylparaben, propylparaben, butylparaben,thymol or mixtures thereof. More preferred preservatives are benzylalcohol, m-cresol, phenol, methylparaben and mixtures thereof. A mostpreferred pharmaceutically acceptable preservative is m-cresol.

The final step in the process of preparing a stock GLP-1 suspension isan adjustment to a final pH between about 6.0 and about 8.5, andpreferably between about pH 6.5 and about pH 8.0, and more preferablybetween about pH 7.0 and about pH 8.0. Although any of a wide variety ofacidification and/or alkalization reagent solutions may be employed forthis pH adjustment, dilute HCl, dilute NaOH and dilute acetic acid arepreferred. More preferred reagent solutions are dilute HCl and diluteNaOH. The preferred pH to which the composition is adjusted will dependto some extent upon the selected peptide, the peptide concentration, theproposed route of administration and the selected buffer.

Preferably, with TRIS as the selected buffer, the pH will be adjusted toa pH between about 6.5 and about 8.5. More preferably, the pH will beadjusted to a pH between about 7.0 and about 7.8, between about 7.2 andabout 7.8, between about 7.5 and about 8.5, or between about 7.0 andabout 8.0. A most preferred pH to which the stock GLP-1 suspension isadjusted when TRIS is the selected buffer is about 7.5. With maleate asthe selected buffer, the pH will be adjusted to a pH between about 6.0and about 7.5. More preferably, the pH will be adjusted to a pH betweenabout 6.4 and about 7.5, between about 6.4 and about 7.0, or betweenabout 6.0 and about 7.0. A most preferred pH to which the stock GLP-1suspension is adjusted when maleate is the selected buffer is about 6.5.

Protamine Complexes:

In another embodiment, the GLP-1 solid phase can be a complex comprisinga GLP-1 compound and a basic polypeptide. Optionally, the complexcomprises a divalent metal ion such as zinc. The complex can be eithercrystalline or amorphous material or a mixture of crystalline andamorphous material. A crystalline complex is comprised primarily ofindividual or clusters of microcrystals, rods, needles, or plates ormixtures thereof. An amorphous complex comprises a precipitate, butlacks matter in a crystalline state and a definable form or structure.Basic polypeptides include but are not limited to basic proteins orpolyamines. Examples of basic proteins or polyamines are polylysine,polyarginine, polyomithine, protamine, putrescine, spermine, spermidine,and histone. Preferred basic polypeptides are polyarginine, protamine,polylysine, and polyornithine. More preferred is polylysine,polyarginine, and protamine. Most preferred is protamine. Protamine isthe generic name of a group of strongly basic proteins present in spermcell nuclei in salt like combination with nucleic acids. Commerciallyavailable protamines can be isolated from mature fish sperm and areusually obtained as the sulfate. The peptide composition of a specificprotamine may vary depending on which family, genera or species of fishit is obtained from. Protamine from salmon or trout can be separatedinto two, three, or more main fractions of proteins that may beseparated further. The different parent peptides consist of about 30amino acids of which more than 20 are arginines. The average molecularweight of protamine is about 4,300. Commercially available protaminesulfate is approximately 80% protamine.

The complex may be prepared by mixing a GLP-1. compound solution with abasic polypeptide solution. A GLP-1 compound solution is preferably abuffered solution and is prepared by dissolving GLP-1 compound in aselected buffer. Examples of a buffer include but are not limited toTRIS, Glycine, Arginine, and Phosphate. A preferred buffer is TRIS. Theconcentration of buffer should be such that changes in hydrogen ionconcentration that would otherwise occur as a result of chemicalreactions are minimized. The pH of the GLP-1 solution is about pH⁶ toabout pH 10, preferably about pH 7 to about pH 10, more preferably aboutpH 8 to about pH 10, and most preferably about pH 9 to about pH 10. ThepH of the GLP-1 compound solution can be adjusted based on theisoelectric point (pI) of the GLP-1 compound being dissolved to optimizethe amount of GLP-1 compound that will dissolve and remain soluble inthe buffered GLP-1 solution. For example, it is preferable thatVal⁸-GLP-1(7-37)OH be dissolved in a TRIS buffered solution wherein thepH is adjusted to 9.0.

The basic polypeptide solution is preferably a buffered solutionprepared by dissolving a basic polypeptide in a selected buffer.Examples of a buffer include but are not limited to TRIS, Glycine,Arginine, and Phosphate. A preferred buffer is TRIS. The concentrationof buffer should be such that changes in hydrogen ion concentration thatwould otherwise occur as a result of chemical reactions are minimized.The pH of the buffered basic polypeptide solution is about pH 6 to aboutpH 10, preferably about pH 7 to about pH 10, more preferably about pH 8to about pH 10, and most preferably about pH 9 to about pH 10. Theconcentration of basic polypeptide in solution is about 1.0 to about20.0 mg/mL. However, ultimately, the concentration of basic polypeptidewill be such that when the basic polypeptide solution is added to theGLP-1 compound solution the desired ratio of GLP-1 compound to basicpolypeptide is achieved. For example, it is preferable that protamine isdissolved in a Tris buffered solution at a pH of 9.0.

To induce complex formation and reduce adhesion of the complex toreaction vessels, an alcohol selected from the group consisting ofethanol, propanol, isopropanol, and methanol, or mixtures thereof, isadded to either the buffered GLP-1 compound solution, the buffered basicpolypeptide solution, or both solutions. It is preferred that the finalconcentration of alcohol once the buffered GLP-1 compound solution andthe buffered basic polypeptide solution are mixed is between about 0.2and about 10% (volume to volume) (v/v). Most preferred is an ethanolconcentration between about 4% and 5% (v/v).

The complex is prepared by mixing a buffered GLP-1 compound solutionwith a buffered basic polypeptide solution. A suspension of amorphousprecipitate is initially formed. However, if primarily crystallinecomplexes are desired, the suspension is incubated for about 18 to 24hours. Although the temperature of incubation is not critical, it ispreferable that the temperature be between about 5° C. and about 35° C.to avoid denaturation of the peptide and to preserve the crystallinematrix that forms. Preferably, the temperature is about 25° C. Theamount of time and temperature of incubation can be varied depending onwhether amorphous complexes, crystalline complexes, or a mixture ofamorphous and crystalline complexes are desired.

The amount of the GLP-1 solution and the basic peptide solution to bemixed together may be adjusted depending on the concentration of GLP-1compound and basic polypeptide and alcohol in each solution such thatthe ratios of GLP-1 compound to basic polypeptide in the final mixturerange from about 4:1 to about 10:1 (w/w). The final ratio of GLP-1compound to basic polypeptide affects the morphology as well as theultimate yield of the complex. For example, a ratio that generallyresults in crystalline complexes comprised of individual and clusters ofmicrocrystals, rods, needles, plates or mixtures thereof is about 5:1(w/w) (GLP-1 compound:basic polypeptide), whereas a ratio of 4:1 (w/w)(GLP-1 compound:basic polypeptide) additionally results in largerclusters of microcrystals, rods, needles, and plates.

The yield of complex formation at ratios between about 4:1 and about 5:1(w/w of GLP-1 compound to basic polypeptide) is generally near 100%.However, the ratio of GLP-1 compound to basic polypeptide can beincreased to above 5:1 (GLP-1 compound: basic polypeptide) even thoughthis results in a decreased yield. The concentrations of GLP-1 compoundin the GLP-1 solution and basic polypeptide in the basic polypeptidesolution can be adjusted such that the ratios of GLP-1 compound to basicpolypeptide range from about 6:1 to about 10:1 (w/w), and morepreferably from about 7:1 to about 9:1 (w/w)(GLP-1 compounds:basicpolypeptide). The yield of complex formation at these ratios is lessthan 95%, usually less than 90%.

In another embodiment, a divalent metal ion such as zinc is added to thesuspension of GLP-1/protamine complex to improve the yield and changethe solubility properties of complexes. The solubility characteristicsof the complex can be effected depending on the amount of zinc addedrelative to the amount of GLP-1 compound present. Such a method forcontrolling the solubility characteristics is useful because thesolubility characteristics of the complex determine the drug releaserate at the site of delivery. Hence by controlling the solubilitycharacteristics, one can control the pharmacokinetic properties of thedrug. Furthermore, soaking the suspension in a solution of zinc candrive the complex formation to completion.

Zinc is preferably added as a salt. Representative examples of zincsalts include zinc acetate, zinc bromide, zinc chloride, zinc fluoride,zinc iodide and zinc sulfate. The skilled artisan will recognize thatthere are many other zinc salts that also might be used. Preferably,zinc oxide, zinc acetate or zinc chloride is used. A buffered zincsolution at pH of between about 5 and about 6 can be added to thesuspension of GLP-1 compound/basic polypeptide complex. A preferredbuffer for the buffered zinc solution is glycine. Optionally, an acidiczinc solution at pH of between about 1 and about 2 can be added to thesuspension. The preferred final molar ratio of zinc to GLP-1 compound isless than about 2:1. Although the temperature of incubation is notcritical, the suspension is generally incubated in the presence of zincbetween about 18 and about 24 hours at a temperature between about 5° C.and about 25° C.

Insulins:

Insulin peptides can be made by a variety of methods well known in theart such as solid-phase synthetic chemistry, purification of insulinfrom natural sources, recombinant DNA technology, or a combination ofthese methods.

Examples of insulin peptides of the present invention include regularhuman insulin and monomeric insulin analogs. Examples of preferredmonomeric insulin analogs are human insulin wherein proline at position28 of the Beta chain is substituted with aspartic acid, lysine, leucine,valine, or alanine and lysine at position 29 of the Beta chain is lysineor proline (AspB28 or AspB28-ProB29, LysB28 or LysB28-ProB29, LeuB28 orLeuB28-ProB29, ValB28 or ValB28-ProB29, AlaB28 or AlaB28-ProB29);deletion of amino acids 28, 29 and 30 of the Beta chain (des B28-30); ordeletion of amino acid 27 of the Beta chain (des B27). More preferredmonomeric insulin analogs are LysB28-ProB29 and AspB28. Preparations ofvarious monomeric insulin analogs are disclosed in U.S. Pat. Nos.5,474,978, and 700,662, and are herein incorporated by reference.

Other examples of monomeric insulin analogs include derivatives orphysiologically tolerable salts thereof in which asparagine (Asn) inposition B3 of the B chain is replaced by a naturally occurring basicamino acid residue and at least one amino acid residue in the positionsB27, B28 or B29 of the B chain is replaced by another naturallyoccurring amino acid residue, it optionally being possible forasparagine (Asn) in position 21 of the A chain to be replaced by Asp,Gly, Ser, Thr or Ala and for phenylalanine (Phe) in position B1 of the Bchain and the amino acid residue in position B30 of the B chain to beabsent. Preferrably B3 is His, Arg, or Lys. Preferrably B27, B28, or B29is Ile, Asp, or Glu. A preferred monomeric insulin analog of this genusis LysB3-GluB29. Preparations of this genus of monomeric insulin analogsare disclosed in U.S. Pat. No. 6,221,633 and is herein incorporated byreference.

Process of Preparing Biphasic Mixtures

The present invention further provides a process of preparing biphasicmixtures suitable for use in pharmaceutical formulations by mixing aGLP-1 solid with an insulin solution. Insoluble GLP-1 precipitates orcrystals may be added as a solid to a solution containing an insulin, orsolid soluble insulin may be dissolved in a suspension containinginsoluble GLP-1 precipitates and crystals. Alternatively, both theinsoluble GLP-1 precipitates or crystals and the soluble insulin may beadded in any order to a buffered solution. It is preferred that stockGLP-1 suspensions and stock insulin solutions be prepared separately andthen added together at the desired ratio. Preferably, a GLP-1 suspensionis diluted with an insulin solution. The GLP-1 suspension and theinsulin solution must be mixed in such a way that the GLP-1 remains in asolid phase and maintains the sustained pharmacokinetic profile and thatthe insulin remains in the solution phase and maintains the short timeaction characteristic.

Preferably, the process comprises mixing a GLP-1 suspension with aLysB28-ProB29 insulin solution of example 5 at a ratio from about 99:1(w/w) to 10:90 (w/w), more preferably, at a ratio from about 85:15 (w/w)to 15:85 (w/w), and even more preferably at a ratio from about 85:15(w/w) to about 50:50 (w/w).

In another embodiment, the process comprises mixing a GLP-1 suspensionwith an AspB28 insulin solution at a ratio from about 99:1 (w/w) to10:90 (w/w) more preferably, at a ratio from about 85:15 (w/w) to 15:85(w/w), and even more preferably at a ratio from about 85:15 (w/w) toabout 50:50 (w/w).

In another embodiment, the process comprises mixing a GLP-1 suspensionwith a regular human insulin solution at a ratio from about 99:1 (w/w)to 10:90 (w/w), more preferably, at a ratio from about 85:15 (w/w) to15:85 (w/w), and even more preferably at a ratio from about 85:15 (w/w)to about 50:50 (w/w).

In another embodiment, the process comprises mixing Val⁸-GLP-1suspension of example 3 with a LysB28-ProB29 insulin solution of example5 at a ratio from about 99:1 (w/w) to 10:90 (w/w), more preferably, at aratio from about 85:15 (w/w) to 15:85 (w/w), and even more preferably ata ratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the process comprises mixing Val⁸-GLP-1suspension of example 3 with an AspB28 insulin solution at a ratio fromabout 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratio from about85:15 (w/w) to 15:85 (w/w), and even more preferably at a ratio fromabout 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the process comprises mixing Val⁸-GLP-1suspension of example 3 with a regular human insulin solution at a ratiofrom about 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratio fromabout 85:15 (w/w) to 15:85 (w/w), and even more preferably at a ratiofrom about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the process comprises mixing either Exendin-3suspension or Exendin-4 suspension with a LysB28-ProB29 insulin solutionof example 5 at a ratio from about 99:1 (w/w) to 10:90 (w/w), morepreferably, at a ratio from about 85:15 (w/w) to 15:85 (w/w), and evenmore preferably at a ratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the process comprises mixing either Exendin-3suspension or Exendin-4 suspension with an AspB28 insulin solution at aratio from about 99:1 (w/w) to 10:90 (w/w), more preferably, at a ratiofrom about 85:15 (w/w) to 15:85 (w/w), and even more preferably at aratio from about 85:15 (w/w) to about 50:50 (w/w).

In another embodiment, the process comprises mixing either Exendin-3suspension or Exendin-4 suspension with a regular human insulin solutionat a ratio from about 99:1 (w/w) to 10:90 (w/w), more preferably, at aratio from about 85:15 (w/w) to 15:85 (w/w), and even more preferably ata ratio from about 85:15 (w/w) to about 50:50 (w/w).

The process can optionally comprise the additional step of adding abuffer to the GLP-1 suspension, the insulin solution, or the biphasicmixture of GLP-1 and insulin. Preferably the buffer is TRIS, glycine, orboth.

The process can optionally comprise the additional step of adding apreservative to the GLP-1 suspension, the insulin solution, or thebiphasic mixture of GLP-1 and insulin.

The process can optionally comprise the additional step of adding anisotonicity agent to the GLP-1 suspension, the insulin solution, or thebiphasic mixture of GLP-1 and insulin.

The process can optionally comprise the additional step of adding apreservative to the GLP-1 suspension, the insulin solution, or thebiphasic mixture of GLP-1 and insulin.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

EXAMPLE 1

Insulinotropic Activity Determination

A collagenase digest of pancreatic tissue is separated on a Ficollgradient (27%, 23%, 20.5%, and 11% in Hank's balanced salt solution, pH7.4). The islets are collected from the 20.5%/11% interface, washed andhandpicked free of exocrine and other tissue under a stereomicroscope.The islets are incubated overnight in RPMI 1640 medium supplemented with10% fetal bovine plasma and containing 11 mM glucose at 37° C. and 95%air/5% CO₂. The GLP-1 compound to be studied is prepared at a range ofconcentrations, preferably 3 nanomolar to 30 nanomolar in RPMI mediumcontaining 10% fetal bovine plasma and 16.7 mM glucose. About 8 to 10isolated islets are then transferred by pipette to a total volume of 250μl of the GLP-1 compound containing medium in 96 well microtiter dishes.The islets are incubated in the presence of the GLP-1 compound at 37°C., 95% air, 5% CO₂ for 90 minutes. Then aliquots of islet-free mediumare collected and 100 μl thereof are assayed for the amount of insulinpresent by radioimmunoassay using an Equate Insulin RIA Kit (Binax,Inc., Portland, Me.).

EXAMPLE 2

Zinc Crystallization of Val⁸-GLP-1(7-37)OH

Val⁸-GLP-1(7-37)OH was dissolved in about 15 mL of sterile water forinjection at a concentration of about 20 mg/mL. The pH was adjusted toabout 10.8 with NaOH and held at ambient temperature for about 30minutes. To this peptide solution was added 66.8 mg glycine and the pHwas adjusted to about 9.5 with NaOH.

The solution was then pressure filtered through a 0.22 μm Millex®-GV(Millipore Corporation, Waltham, Mass., USA) sterilizing filter membraneunit.

To the stirred, sterile filtrate was added 3.3 mL of a sterile 50%(byvolume) ethanol solution that had been prepared from absolute ethanoland water for injection.

To this stirred solution was added 0.89 mL of a sterile-filteredbuffered zinc oxide solution that had been prepared by combining about1.221 mg zinc oxide, about 12 mL of 10% Hydrochloric acid, about 85 mLwater for injection, and 528.7 mg glycine. The pH of the zinc oxidesolution was adjusted to 5.68 with about 3 to 4 mL of 10% NaOH and thefinal volume was adjusted to 100 mL with water for injection.

The pH of the resulting Val⁸-GLP-1 solution was adjusted to about 9.1with NaOH. After gently mixing for about 5 minutes, the crystallizationsolution was covered tightly and held quiescently at ambient temperaturefor crystallization. At this point the Val⁸-GLP-1 concentration wasdetermined to be about 12.9 mg/mL, glycine concentration was about 50mM, ethanol concentration was about 9% and zinc oxide concentration was7 mM (0.46 mg/mL).

After about 24 hours the crystallization process was complete. Analysestypically showed mostly thin, plate-like crystals in a yield greaterthan 98%.

EXAMPLE 3

Preparing the Stock Val⁸-GLP-1 Suspension.

To 15 mL of the completed crystallization suspension described inExample 2 was added, with stirring, 15 mL of a sterile-filtered solutioncomprising about 30 mM TRIS, 220 mM NaCl, and 6 mg/mL m-cresol at aboutpH 7.5.

The stable suspension prepared as described above comprised about 6.5mg/mL of thin, plate-like crystals of Val⁸-GLP-1(7-37)OH, about 24.8 mMglycine, about 3 mg/mL m-cresol, about 110 mM sodium chloride, about4.3% ethanol by volume, about 0.23 mg/mL zinc, about 15 mM TRIS and hasa pH of about 7.5.

EXAMPLE 4

Preparation of Insulin

LysB28-ProB29 insulin was dissolved in about 15 mL of 0.01N HCl at aconcentration of about 200 U/mL. To this solution, 68 mg of glycine wasadded and the pH was adjusted to 7.5 with NaOH. The resulting solutionwas pressure filtered through a 0.22 μm Millex®-GV (MilliporeCorporation, Waltham, Mass., USA) sterilizing filter membrane unit.

EXAMPLE 5

Preparing the Stock LysB28-ProB29 Insulin Solution.

To 15 mL of the LysB28-ProB29 insulin solution described in Example 4was added, with stirring, 15 mL of a sterile-filtered solutioncomprising about 30 mM TRIS, 220 mM NaCl, and 6 mg/mL m-cresol at aboutpH 7.5.

The stable pharmaceutical solution prepared as described above comprisedabout 100 U/mL of LysB28-ProB29 insulin, about 30 mM glycine, about 3mg/mL m-cresol, about 110 mM sodium chloride, about 15 mM TRIS and has apH of about 7.5.

EXAMPLE 6

Val⁸-GLP-1 Suspension/LysB28-ProB29 Insulin Solution Mixtures

The Val⁸-GLP-1 suspension of example 3 was mixed with the LysB28-ProB29insulin solution of example 5 at two different ratios.

Mixuture A (85:15, GLP-1:insulin, weight:weight) was prepared by mixing15 mL of stock Val⁸-GLP-1 suspension with 5 mL of stock LysB28-ProB29insulin solution. The resulting mixture comprised about 4.9 mg/mL (1.4mM) Val⁸-GLP-1, about 25 U/mL LysB28-ProB29 insulin, about 26 mMglycine, about 0.17 mg/mL zinc, about 15 mM TRIS, about 110 mM sodiumchloride, about 3.2% ethanol by volume, about 3 mg/mL m-cresol, and hasa pH of about 7.5.

Mixuture B (38:62, GLP-1:insulin, weight:weight) was prepared by mixing5 mL of stock Val⁸-GLP-1 suspension with 15 mL of stock LysB28-ProB29insulin solution. The resulting mixture comprised about 1.6 mg/mL (0.48mM) Val⁸-GLP-1, about 75 U/mL LysB28-ProB29 insulin, about 29 mMglycine, about 0.06 mg/mL zinc, about 15 mM TRIS, about 110 mM sodiumchloride, about 1.1% ethanol by volume, about 3 mg/mL m-cresol, and hasa pH of about 7.5.

EXAMPLE 7

Stability Studies of the Mixtures

The mixtures described in example 6 were tested for stability at two andfour weeks at 5° C. and 30° C. The volume diameter distribution wasdetermined using a laser light scattering particle analyzer such asCoulter LS 230. (Coulter Electronics Limited, Luton, Beds, England). Thesamples were also analyzed by reversed-phase HPLC chromatography todetermine the total protein concentrations and the soluble proteinconcentrations. The data are summarized below.

Mixture A at 5° C.

Size measured by Coulter:

V mean (μm) 10% < (μm) 50% < (μm) 90% < (μm) Initial 7.61 1.84 7.15 14.12 weeks 7.50 1.89 6.98 13.8 4 weeks 7.33 1.77 6.84 13.5Val⁸-GLP-1 HPLC analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 5.14 0.012 0.003 2 weeks 4.69 0.020 0.014 4 weeks 5.02 0.0200.022LysB28-ProB29 Insulin HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 0.98 0.95 0.85 2 weeks 0.90 0.96 0.87 4 weeks 0.92 0.84 0.88Mixture A at 30° C.Size Measured by Coulter:

V mean (μm) 10% < (μm) 50% < (μm) 90% < (μm) Initial 7.61 1.84 7.15 14.12 weeks 8.02 1.96 7.53 14.7 4 weeks 7.25 1.72 6.82 13.4Val⁸-GLP-1 HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 5.14 0.012 0.003 2 weeks 4.95 0.016 0.130 4 weeks 5.40 0.0150.034LysB28-ProB29 Insulin HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 0.98 0.95 0.85 2 weeks 0.94 0.97 0.90 4 weeks 0.86 0.86 0.94Mixture B at 5° C.Size measured by Coulter:

V mean (μm) 10% < (μm) 50% < (μm) 90% < (μm) Initial 7.61 1.84 7.15 14.12 weeks 7.50 1.82 7.07 13.8 4 weeks 7.72 1.75 6.98 13.6Val⁸-GLP-1 HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 1.71 0.014 0.002 2 weeks 1.82 0.020 0.043 4 weeks 1.66 0.0180.019LysB28-ProB29 Insulin HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 2.59 2.60 2.52 2 weeks 2.63 2.72 2.60 4 weeks 2.56 2.46 2.65Mixture B at 30° C.Size measured by Coulter:

V mean (μm) 10% < (μm) 50% < (μm) 90% < (μm) Initial 7.61 1.84 7.15 14.12 weeks 8.10 1.94 7.70 14.8 4 weeks 7.29 1.70 6.85 13.4Val⁸-GLP-1 HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 1.71 0.014 0.002 2 weeks 1.98 0.019 0.180 4 weeks 1.88 0.0150.017LysB28-ProB29 Insulin HPLC Analysis:

Immediately Total potency Available Supernatant (mg/mL) (mg/mL) (mg/mL)Initial 2.59 2.60 2.52 2 weeks 2.78 2.86 2.74 4 weeks 2.59 2.75 2.78

EXAMPLE 8

In Vivo Studies

Val⁸-GLP-1 Suspension/LysB28-ProB29 Insulin Solution Mixtures arePrepared as Described in Example 6.

The mixture is injected into a single site such that 0.74 U/kgLysB28-ProB29 insulin (1.5 nmol/kg Val⁸-GLP-1 suspension) isadministered. A 3-hour hyperglycemic (150 mg/dl) clamp is initiated andglucose infusion rates are continually recorded. Blood samples are takenperiodically for the determination of plasma glucose, insulin,C-peptide, and immunoreactive GLP-1 concentrations. Plasma glucoseconcentrations are determined on the day of study. The remainder of thesamples are then frozen (−80° C.) and assayed for hormone concentrationdeterminations at a later time.

1. A pharmaceutical formulation comprising a biphasic mixture whichcomprises a GLP-1 compound in a solid phase and an insulin in a solutionphase.
 2. The pharmaceutical formulation of claim 1 wherein the GLP-1compound has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ IDNO:
 6. 3. The pharmaceutical formulation of claim 1 wherein the insulinis selected from the group consisting of; AspB28, AspB28-ProB29, LysB28,LysB28-ProB29, LysB3-GluB29, LeuB28, LeuB28-ProB29, ValB28,ValB28-ProB29, AlaB28, AlaB28-ProB29, des(B28-B30)-human insulin; anddes (B27)-human insulin.
 4. The pharmaceutical formulation of claim 3wherein the insulin is AspB28.
 5. The pharmaceutical formulation ofclaim 3 wherein the insulin is LysB28-ProB29.
 6. The pharmaceuticalformulation of claim 1 wherein the GLP-1 compound has a sequence of SEQID NO: 2 or SEQ ID NO: 3 and the insulin is AspB28, LysB28-ProB29, orLysB3-GluB29.